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Dear Editor,
Listeria monocytogenes is a Gram‐positive, non‐sporeforming, facultatively anaerobic bacilli that can commonly isolated from various animals, humans, and environment. It is pathogenic to humans and often regarded as a paradigm for intracellular parasitism. 1 Due to its widespread distribution, L. monocytogenes has many opportunities to enter human food processing environment, resulting in contamination of food production. 2 , 3 Infection of humans ingesting colonized food is potentiated by the ability of the organism to multiply at 4°C. As a food‐borne pathogen, L. monocytogenes primarily causes febrile gastroenteritis, and in severe cases can also cause septicemia, meningitis, and central nervous system infection with a high mortality. 4 , 5 , 6 L. monocytogenes caused blood stream infection has been reported sporadically worldwide. However, genomic and phylogenetic feature of L. monocytogenes strains causing septicemia are not fully understood. Here, we report the genome sequence of a clinical L. monocytogenes strain isolated from a blood stream infection of an immunocompetent patient in China. Genotypic characterization and phylogenetic analysis of this strain were further conducted.
L. monocytogenes strain CDHLST was isolated from a blood sample of a 41‐year‐old male patient who hospitalized in the department of infectious disease in a teaching hospital in Zhejiang Province and diagnosed with septicemia in 2019. The patient was physically fit in the past, without history of hypertension, diabetes, infectious diseases, allergies, or surgery except for alcoholic hepatitis and alcohol withdrawal syndrome. Initial routine blood test and procalcitonin (PCT) were as follows: white blood cells (WBCs), 10.91 × 109/L (neutrophils 89.2%, lymphocytes 6.4%); red blood cell, 3.91 × 1012/L and PCT, 0.25 ng/ml. Three days later after his hospitalization, result of routine blood test and PCT were changed to white blood cells (WBCs), 13.70 × 109/L (neutrophils 86.7%, lymphocytes 9.1%); red blood cell, 3.57 × 1012/L and PCT, 1.89 ng/ml. Bacterial culture of blood samples suggested a L. monocytogenes septicemia. Ampicillin/Sulbactam was used for antibacterial treatment and the patient improved after a week of treatment.
The isolate was identified using the VITEK MS system (bioMérieux) and conducted for whole genome sequencing using the Illumina NovaSeq 6000 platform (Illumina Inc.,). The whole genome sequence was de novo assembled using SPAdes 3.13.0 and automatically annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) server. Multilocus sequence typing (MLST), antimicrobial resistance genes (ARGs), and virulence genes (VGs) of the isolate were analyzed using the BacWGSTdb server. 7 , 8 , 9 Phylogenetic relationship between CDHLST and other L. monocytogenes strains was analyzed using a core genome multilocus sequence typing (cgMLST) approaches with threshold 25 for phylogenetic analysis. 10 The cgMLST scheme is based on whole genome sequencing and over 2,000 core genome genes are included, which is essentially different from the conventional seven gene MLST strategy and ideally suited to standardize whole genome sequencing based bacterial genotyping. Antimicrobial susceptibility testing was conducted using the Etest method following the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Penicillin, ampicillin, trimethoprim/sulfamethoxazole, and meropenem were used in the test.
L. monocytogenes strain CDHLST was resistant to penicillin, but susceptible to ampicillin, trimethoprim/sulfamethoxazole, and meropenem. According to the in silico MLST analysis, CDHLST belongs to sequence type (ST) 87. The whole genome sequence of CDHLST consists of 17 contigs that comprised 2,943,808 bp with a G + C content amounted to 37.9%. A total of 2,871 protein‐coding sequences, 6 rRNA genes, 54 tRNA genes and 4 ncRNA operons were identified by the PGAP server. The genome contains one CRISPR sequence (CRISPR length: 2544 bp, located in contig00003, position: 360305–362849) and several IS elements, the majority of which belong to the IS3, IS4, and IS1595 families. The ARGs and VGs of CDHLST are presented in Table 1. One fosfomycin resistance gene, fosX, and thirty virulence genes including actA, ami, aut, bsh, clpC, clpE, clpP, fbpA, gtcA, and hly were identified in CDHLST.
TABLE 1.
Antimicrobial resistance genes and virulence genes identified in L. monocytogenes strain CDHLST
| Antimicrobial resistance gene | Contig | Identity (%) | Position | Antimicrobial resistance category |
|---|---|---|---|---|
| fosX | contig00009 | 92.56 | 17604..18005 | fosfomycin |
| Virulence gene | Contig | Identity (%) | Position | Functional annotation |
| actA | contig00008 | 94.1 | 129255..131169 | actin‐assembly inducing protein precursor |
| ami | contig00007 | 97.93 | 204526..207279 | autolysin amidase, adhesin |
| aut | contig00001 | 96.97 | 183609..185327 | autolysin |
| bsh | contig00004 | 97.55 | 44831..45808 | bile salt hydrolase |
| clpC | contig00006 | 94.11 | 2288..4750 | endopeptidase Clp ATP‐binding chain C |
| clpE | contig00001 | 93.38 | 257886..260054 | ATP‐dependent protease |
| clpP | contig00007 | 96.82 | 98739..99335 | ATP‐dependent Clp protease proteolytic subunit |
| fbpA | contig00005 | 91.77 | 202054..203766 | fibronectin‐binding protein |
| gtcA | contig00007 | 98.63 | 194341..194778 | wall teichoic acid glycosylation protein GtcA |
| hly | contig00008 | 97.23 | 125599..127188 | listeriolysin O precursor |
| hpt | contig00001 | 97.69 | 416141..417526 | hexose phosphate transport protein |
| iap/cwhA | contig00001 | 95.87 | 665958..667391 | P60 extracellular protein, invasion associated protein |
| inlA | contig00006 | 96.55 | 218554..220956 | Internalin A |
| inlB | contig00006 | 94.98 | 221041..222931 | Internalin B |
| inlC | contig00005 | 97.08 | 246307..247197 | internalin C |
| inlJ | contig00003 | 95.07 | 113531..116071 | internalin J |
| inlK | contig00002 | 89.18 | 427325..429145 | internalin K |
| inlP | contig00007 | 95.29 | 100992..102158 | internalin P |
| lap | contig00002 | 97.27 | 65697..68297 | Listeria adhesion protein Lap |
| lapB | contig00002 | 93 | 20677..25833 | Listeria adhesion protein LapB |
| lpeA | contig00005 | 96.89 | 183162..184094 | lipoprotein promoting cell invasion |
| lplA1 | contig00001 | 92.77 | 317149..318144 | lipoate protein ligase |
| lspA | contig00005 | 97.42 | 187019..187483 | signal peptidase II |
| mpl | contig00008 | 94.72 | 127523..129055 | Zinc metalloproteinase precursor |
| oatA | contig00002 | 93.59 | 425406..427292 | peptidoglycan O‐acetyltransferase |
| pdgA | contig00006 | 97 | 203941..205341 | Peptidoglycan N‐deacetylase |
| plcA | contig00008 | 97.27 | 124403..125356 | phosphatidylinositol‐specific phospholipase c |
| plcB | contig00008 | 95.51 | 131211..132079 | phospholipase C |
| prfA | contig00008 | 97.48 | 123417..124130 | listeriolysin positive regulatory protein |
| prsA2 | contig00004 | 96.15 | 210112..210993 | post translocation chaperone PrsA2 |
Phylogenetic relationship between L. monocytogenes strain CDHLST and other L. monocytogenes strains currently deposited in the BacWGSTdb 2.0 server was presented in Figure 1. The database currently contains 3243 L. monocytogenes strains. A total of 60 phylogenetically related strains were identified in the database according to a threshold of 25 and the clinical metadata of these isolates are presented in Table S1. Among them, 54 strains were from China, four strains from the USA and two strains of unknown location. The 54 strains from China were distributed in 11 provinces, of which Sichuan, Beijing and Zhejiang were the most predominant. The closest relatives of L. monocytogenes strain CDHLST are two L. monocytogenes strain isolated from Hebei in 2005 and 2006, ICDC_LM0200 and ICDC_LM0216 (Accession No. QDCN00000000 and QDCL00000000); the sequences differ by only 9 cgMLST loci and bearing the same ARGs and VGs. The in silico MLST analysis showed that all strains, except one ST310 strain and one ST1166 strain, belonged to ST87. These ST87 L. monocytogenes strains were epidemiologically related and have spread over different provinces in China. They have similar ARGs and VGs, and widely distributed in poultry, aquatic product, meat, cooked food, environment, feces, and even can be isolated from umbilical cord blood (Table S1).
FIGURE 1.
Phylogenetic relationship between CDHLST and other L. monocytogenes strains currently deposited in the BacWGSTdb 2.0 server using a core genome multilocus sequence typing (cgMLST) approaches. The cells in different shape indicate a distinct of ST type, while the color of each cells indicates a distinct of isolate location
In summary, we report the first genome sequence of a clinical ST87 L. monocytogenes strain isolated from a blood stream infection of an immunocompetent individual in China. Our data may help to understand the genomic features and phylogenetic characterization of this bacterial pathogen.
This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number JAFIQZ000000000.
Supporting information
Table S1
ACKNOWLEDGEMENTS
This study was supported by grants from the National Natural Science Foundation of China (81702042), Natural Science Foundation of Zhejiang Province (LQ19H200003), Natural Science Foundation of Zhejiang Provincial Department of Education (Y202044471) and Zhejiang Provincial Medical and Health Science and Technology Plan (2019KY311).
Weizhong wang and Juan Xu contributed equally to this study.
DATA AVAILABILITY STATEMENT
The data presented in the study are deposited in the GenBank repository, accession number JAFIQZ000000000. Interested readers can contact the corresponding author for further information.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1
Data Availability Statement
The data presented in the study are deposited in the GenBank repository, accession number JAFIQZ000000000. Interested readers can contact the corresponding author for further information.